Use Image > Adjust > Brightness/Contrast on individual channels via the Color > Channels Tool .
When comparing staining or fluorescence, measuring intensity (gray values) is critical.
Perfect for comparing two channels or a before/after state: imagej image comparison
ImageJ will create a new image where identical areas turn grey. Areas that changed will flash into high-contrast colors (depending on your LUT). This is excellent for spotting a missing cell in a time-lapse or an altered background in a photo.
Now, open your images, run the Image Calculator, and let the pixels tell you the truth. Use Image > Adjust > Brightness/Contrast on individual
Subtraction tells you how much things changed. The Difference mode tells you where .
The golden rule of image comparison is: For pixel-wise operations, your two images must be perfectly aligned. If they are misaligned by even one pixel, a subtraction will produce artifacts resembling edges rather than real differences. Areas that changed will flash into high-contrast colors
For high-throughput comparison (e.g., 100 image pairs), use ImageJ's macro language.